Biological Characteristics of HaCaT Cells Expressing Exogenous hEGF Gene and Induced Function of Skin Sppendage Cell Transformation
|School||Fourth Military Medical University|
|Keywords||ESC desmocyte adipose-derived stem cells epidermal growthfactor rat tail collagen tissue-engineered skin sweat gland|
【Background】Due to lack of donor site in extensive deep burns, skin defect area can not beregenerated , and leave severe disablility to patients. For this reason, people hasbeen searching for a long time to solve the shortage of skin source in large-scaleburn, but the traditional methods such as: auto reticulate skin, large sheet ofheterogeneous skin embedded window autologous micro skin graft, particulateskin, and autologous transplantation of epidermal cell culture amplification,although have solved some problems to some extent, but these methods face acommon deficiency: different degrees of scar formation cause poor appearance,serious deformities cause dysfunction and so on [2-5]. In this regard, people beganto construct tissue engineering of skin in the hope to solve this problem in recentyears, from the earliest design of artificial dermis by Burk and Yannas to thecurrent U.S. FDA approval for clinical use of living cells containing the human skin substitute Apligraf development, which proved the feasibility of constructingtissue engineered skin, although these products are also facing some drawbacks:the lack of sweat glands and hair follicles and other cutaneous appendages(adnexa) and the difficulty of obtaining appearance and physiological function ofnormal skin .There is no hair in extensive deep burn wound repair, no sweatglands in particular, which affects not only Appearance, but also the quality oflife after wound healing. Therefore, reconstruction of skin appendage has been toexplore the direction of the field of skin tissue repair. We take epidermal stemcells (ESC) as seed cells, rat tail collagen as a scaffold, epidermal growthfactor(secreted by HaCaT cells transfected by hEGF gene in our laboratory) withsignificant induction effect for the formation of sweat glands and hair follicles ofskin appendages, as well as dermal fibroblasts and adipose-derived stem cells, asthe ESC proliferation and differentiation factors, then construct tissue engineeredskin to explore the possibility of human ESC differentiation into sweat glands,hair follicles of skin appendage, and analysis its differentiation mechanism.【Objective】1. To study the HaCaT cells transfected by constructed expression plasmidpcDNA3.1-hEGF in our laboratory, and observe the Biological characteristics:morphology, proliferation, secretion of EGF immune phenotype.2. To explore efficient Isolation and culture method of human ESC, observe thebiological characteristics, and identify morphology, immunophenotype, etc.3. To construct the skin tissue engineering, explore different methods andconditions on the construction of the biological effects of tissue-engineeredskin.4. Making use of cell culture model in vitro for skin dermal (stem) cells combined with cellularized derma, carry on exploratory study on In vitrophenotype translation of skin appendage.【Research】1. Observe and identify morphology, proliferation, molecular phenotype of theHaCaT cells transfected by constructed expression plasmid pcDNA3-hEGF inour laboratory.2. Isolate and culture ESC, observe the morphological characteristics, measuregrowth curves and identify molecular phenotype.3. Isolate and culture ADSCs, observe morphology, osteogenic and adipogenicdifferentiation, identify the molecular phenotype by flow cytometry.4. Etablish a stable three-dimensional tissue engineering skin, take EGF as agrowth factor to its biological effects.5. Make use of exogenous EGF to induce ESC complex tissue engineeringderma transformation into the sweat glands.【Results】1. hEGF-transfected HaCaT cells secreted high levels of EGF. HaCaT cellsstably transfected with EGF gene differentiated relevant markers K19,integrin-β1 expression increased in molecular phenotype identification.2. It was available to obtain a large number of higher activity ESC making useof improved isolation and cultivation methods. And we took advantage ofmolecular phenotype to identify the expression of phenotypic K19 andintegrin-β1.3. After ADSCs isolation and culture, we measured CD29, CD90, CD105positive expression; CD31, CD43, CD45 negative expression by flow cytometry, ADSCs showed the Phenotype characteristics of bone cells, fatcells of through induced differentiation.4. To ESC as seed cells, rat tail collagen as a scaffold, EGF as a growth factorin building three-dimensional tissue engineering of skin, tissue sectionsshowed that it Differentiated into three-tier, with the basic characteristics ofskin tissue.5. By RT-PCR, the sweat gland cell surface antigen CK19 and CEA’s mRNAlevels rised in the tissue engineered skin.【Conclusions】1. hEGF-transfected HaCaT cells have highly efficient and stable secretion ofEGF，and show general biological characteristics of epidermal cells throughmolecular phenotype identification.2. It is available to obtain a large number of higher activity ESC making use ofimproved isolation and cultivation methods, with the general biologicalproperties.3. Enzyme digestion can effectively purify human ADSCs, cell grow stably,proliferate actively, ADSCs has the general biological characteristics.4. It was available to make take ESC as seed cells, rat tail collagen as a scaffold,EGF as a growth factor for constructing the three-dimensional tissueengineering skin, its structure close to the physiological skin structure.5. EGF can induce ESC complex tissue engineering derma transformation intothe skin appendage (sweat glands), and plays a decisive role.